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Am J Physiol Heart Circ Physiol 281: H596-H605, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H596-H605, August 2001

Inotropic response of rabbit ventricular myocytes to endothelin-1: difference from isolated papillary muscles

M. A. Hassan Talukder, Ikuo Norota, Kiyoharu Sakurai, and Masao Endoh

Department of Pharmacology, Yamagata University School of Medicine, Yamagata 990-9585, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Endothelin-1 (ET-1) increased cell shortening and Ca2+ transients over the concentration of 3 × 10-11 M to 10-9 M with EC50 of 8.3 × 10-11 M in rabbit single ventricular myocytes. Thus ET-1 was approximately 60 times more potent in single myocytes than in papillary muscles (EC50 = 5.1 × 10-9 M) of the same species. In single myocytes, ET-1 at 10-8 M elicited an inhibitory response that counteracted the facilitatory response: the concentration-response curve (CRC) for ET-1 was bell shaped. The ETA-receptor antagonist BQ-485 shifted CRC for ET-1 to the right in parallel; however, the facilitatory response to 10-8 M ET-1 was markedly enhanced by BQ-485 and also by the ETB antagonist BQ-788. The ETA/ETB antagonist TAK-044 abolished the ET-1-induced response. These findings indicate that the response to ET-1 of single myocytes is different from that of papillary muscles in concentration dependence, characteristics of the response, and susceptibility to ET-receptor antagonists. Anomalous pharmacological characteristics of ET-1-induced response in rabbit papillary muscles may be due to integrated regulatory mechanisms that may involve also various types of noncardiac cell in ventricular myocardium.

adult rabbit cardiomyocytes; cell shortening; Ca2+ transients; ETA receptor; ETB receptor


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ENDOTHELIN-1 (ET-1) is produced by various types of cardiovascular cells, including endothelial cells, vascular smooth muscle, and myocardial cells, and released into the circulating blood in a number of cardiovascular diseases such as congestive heart failure, myocardial infarction, atherosclerosis, and hypertension (15). It is, therefore, postulated that ET-1 may play a crucial role as an autocrine and/or paracrine regulator of cardiovascular function under various cardiovascular disorders (11, 13).

ET-1 elicits a pronounced positive inotropic effect (PIE) in mammalian cardiac muscle of most species, including rat, guinea pig, ferret, rabbit, and human (1, 18, 20, 21, 24). The PIE of ET-1 was most pronounced in the rabbit among mammalian species examined (24). The PIE of ET-1 in the rabbit papillary muscle showed atypical pharmacological characteristics that could not be explained by conventional classification of ET receptor subtype (12). ET-1 and ET-3 elicited the PIE essentially with equivalent efficacy and potency in the rabbit papillary muscle (24). Whereas the PIE of ET-3 was highly susceptible to the selective ETA-receptor antagonists (2), the main portion of the concentration-response curve (CRC) for the PIE of ET-1 was resistant to the conventional selective and nonselective ET-receptor antagonists, such as BQ-123, FR-139317, RES-701-1, IRL-1620, and PD-145065 (5, 12, 19). It is postulated therefore that divergent factors, including neurotransmitters and endogenous regulators such as cytokines, peptides and nitric oxide released from various types of cells in multicellular ventricular muscle preparations and/or the diffusion, which is a limiting factor due to endocardial endothelium in papillary muscle preparations, may contribute to the anomalous pharmacological characteristics of the inotropic response to ET-1 in the rabbit papillary muscle.

The present study was undertaken to characterize the inotropic response of single rabbit ventricular cardiomyocytes to ET-1 to elucidate whether the pharmacological characteristics differ from those in isolated rabbit papillary muscles (multicellular preparation). It was found that the inotropic response to ET-1 and the effectiveness of ET-receptor antagonists in single ventricular myocytes are quite different from those in multicellular preparation in respect to concentration dependence, mode of the response, and pharmacological characteristics.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The current study involving experimental animals conforms to the institutional standards. The study was performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Approval for the animal experiments was obtained from the Committee of Animal Experimentation, Yamagata University School of Medicine, before the experiments, and the study also was carried out in accordance with the Declaration of Helsinki.

Isolation of ventricular cardiomyocytes from rabbit heart. Single ventricular cardiomyocytes were isolated by the Langendorff procedure (7) with a slight modification. Briefly, adult male Japanese White rabbits (1.8-2.0 kg) were anesthetized with pentobarbital sodium (50 mg/kg iv) and given heparin (500 U/kg iv). The heart was quickly removed and immediately attached to an aortic cannula of a modified Langendorff apparatus for perfusion. The continuous retrograde perfusion with HEPES-Tyrode solution was started at a perfusion pressure of 80 cmH2O to washout the blood in the heart for approximately 1 min. The HEPES-Tyrode solution contained (in mM) 136.5 NaCl, 5.4 KCl, 0.53 MgCl2, 1.8 CaCl2, 0.33 NaH2PO4, 5.0 glucose, and 5.0 HEPES (pH 7.4 adjusted with NaOH). The solution was continuously gassed with 100% O2 at 37°C. The heart was then perfused with nominally Ca2+-free HEPES-Tyrode solution containing 0.6 mg/ml collagenase (Type II, Worthington Biochemical; Freehold, NJ) and 0.1 mg/ml protease (Type XIV, Sigma; St. Louis, MO). After ~20 min, when the heart became homogeneously soft, the enzymes were washed out for 1 min by perfusion with HEPES-Tyrode solution containing 0.2 mM CaCl2. The ventricles were then removed, cut into small pieces, placed in HEPES-Tyrode solution containing 0.2 mM CaCl2, and shaken gently on a shaker for easy dissociation of cells. The dispersed cells were filtered through a nylon-mesh (200 µm), and the cell suspension was rinsed several times with gradual increase of Ca2+ in HEPES-Tyrode solution in a stepwise manner up to 1.8 mM. Cells were finally suspended in HEPES-Tyrode solution containing 1.8 mM CaCl2 and kept at room temperature (25 ± 1°C) for 1 h or longer until they were used for experiments.

Indo 1 loading, cell superfusion, and electrical stimulation. Myocytes were loaded with an acetoxymethyl ester form of Ca2+ fluorescent probe, indo 1 (indo 1-AM), by incubating with 5 µM indo 1-AM solution in HEPES-Tyrode buffer for 1-4 min at room temperature. All of the following experimental steps were then carried out at room temperature (25 ± 1°C). After loading, the myocytes were layered onto HEPES-Tyrode buffer in a superfusion chamber on the stage of an inverted microscope (Diaphot TMD300, Nikon; Tokyo, Japan) equipped for simultaneous recording of cell length and indo 1 fluorescence, and allowed to settle down by gravity for 15 min. Continuous superfusion was then started with Krebs-Henseleit bicarbonate buffer at a rate of 2 ml/min, and 10 min later myocytes were stimulated electrically with square-wave pulses of 5 ms duration by bipolar platinum electrodes placed in the perfusion chamber at 0.5 Hz with voltage about 50% above the threshold. After an equilibration period of 40 min under electrical stimulation, the experimental protocols were carried out at room temperature. The cells used were rod shaped with clear transverse striations and had no blebs, no granulation, and no spontaneous contraction. Cells having only stable baseline contractile amplitude with a clear fluorescence ratio after electrical stimulation were used for the experiments. The bicarbonate buffer contained (in mM) 116.4 NaCl, 5.4 KCl, 0.8 MgSO4, 1.8 CaCl2, 1.0 NaH2PO4, 23.8 NaHCO3, 5.0 glucose; the buffer was continuously gassed with 95% O2-5% CO2 (pH 7.4).

Simultaneous measurements of cell shortening and indo 1 fluorescence ratio. Indo 1 fluorescence was excited with the light from a xenon lamp (150 W) with a wavelength of 355 nm, reflected by a 380-nm long-pass dichroic mirror and detected by means of a fluorescence spectrophotometer (CAM-230, Japan Spectroscopic; Tokyo, Japan). Excitation light was applied to the myocytes through a neutral density filter to minimize the photobleaching of indo 1. The emitted fluorescence was collected by an objective lens (CF Fluor DL40; Nikon) and after passing through a 380-nm long-pass dichroic mirror (Omega Optical; Brattleboro, VT), it was separated by a 580-nm long-pass dichroic mirror (Omega Optical). The fluorescence light was subsequently split by a 425-nm dichroic mirror to permit simultaneous measurements of both 405-nm and 500-nm wavelengths through bandpass filters, respectively, by the use of two separate photomultiplier tubes. The fluorescence ratio (405:500 nm) was used as an index of intracellular Ca2+ concentration ([Ca2+]i).

The cell length was monitored simultaneously with indo 1 fluorescence using red light (>620 nm) through the normal bright-field illumination optics of the microscope. The bright-field image of the cell was collected first by a 580-nm long-pass dichroic mirror. The bright-field image of a cell was projected onto a photodiode array of an edge detector (C6294-01, Hamamatsu Photonics; Hamamatsu, Japan) and scanned at every 5 ms. In the current study, an increase or decrease in cell shortening is considered to reflect qualitatively the PIE or negative inotropic effect (NIE) in isometric contractions, and is often referred to as PIE or NIE interchangeably without explanation.

Data recording and analysis. Cell length and indo 1 fluorescence signals were collected in a Macintosh Computer (Power Macintosh 8100/100 AV, Apple Computer; Cuptertino, CA) by means of an A/D Converter (MP-100A, BIOPAC Systems; Santa Barbara, CA) at 200 Hz. Signals were analyzed after low-pass filtering (25-Hz cutoff frequency) and after averaging five successive signals.

Experimental values are presented as means ± SE. The EC50 value for ET-1 was obtained by graphic analysis of mean CRCs for both cell shortening and indo 1 ratio. For statistical analysis of multiple measurements obtained from a single preparation, we used one-way analysis of variance for repeated measures with Bonferroni test. The mean values between two groups were compared by Student's t-test for unpaired values. A P value <0.05 was judged to indicate a statistically significant difference.

Study protocol for cell shortening and [Ca2+]i. After an equilibration period of 40 min, myocytes were exposed to the solution containing different concentrations of ET-1. ET-1 was administered either in a cumulative manner or by single administration. To determine the CRC for ET-1, the superfusate was switched to a solution that contained a higher concentration of ET-1 when the effects of the previous concentration reached a steady level. Where the influence of different ET antagonists were investigated, the antagonists were allowed to act for 20 min before the administration of the agonist and were present in the superfusate throughout the experiments. ET-1 was administered only once to each myocyte because the response to ET-1 was not reversed by washout. The cell length was monitored continuously throughout the experiments, whereas the indo 1 fluorescence was monitored only intermittently to reduce the potential photobleaching. Simultaneous recordings were made at the baseline state and in the presence of the agonist and/or antagonist when the response reached a steady level. The CRC for ET-1 determined by cumulative administration served as the control for examination of the influence of BQ-485, whereas the response to ET-1 administered by the single administration allowed us to investigate the time course of the response after the administration of ET-1 at various concentrations.

Papillary muscle preparation and experimental protocols. Details for experimental procedures used for isolated rabbit right ventricular papillary muscle preparation have been described previously (27). Briefly, two or three papillary muscles (<1 mm in diameter, ~5 mm in length) were isolated from the right ventricle of each rabbit and mounted vertically in 20-ml organ baths that contained Krebs-Henseleit solution (with 0.057 mM ascorbic acid and 0.027 mM EDTA-2Na). The solution was bubbled constantly with 95% O2-5% CO2 at 37°C (pH 7.4). Muscles were electrically stimulated by square-wave pulses of 5-ms duration at a voltage approximately 20% above the threshold, at a frequency of 1 Hz, through bipolar platinum electrodes. The developed tension was recorded on a thermal pen-writing oscillograph (Recti-Horiz-8K, NEC San-ei Instruments; Tokyo, Japan) by means of force-displacement transducers (Shinkoh UL 10 GR, Minebea; Nagano, Japan). The muscle was equilibrated for 60 min in drug-free solution. During the equilibration period, the muscles were stretch initially at a resting tension of 5 mN, and the length was later adjusted to give 90% of the maximal contractile force (Lmax). The concentration of ET-1 in the organ bath was increased in a cumulative manner in steps of 0.5 log units. When steady contractile force had been achieved, ET-1 was added to yield the next higher concentration. The effect of ET-1 was considered to be maximal when the successive concentrations failed to produce a further increase in contractile force.

Drugs and chemicals. The following drugs were used: ET-1 and ET-3 (Peptide Institute; Osaka, Japan), isoproterenol hydrochloride and protease (Type XIV, Sigma), collagenase (Type II, Worthington Biochemical), indo 1-AM (Dojin Chemical; Kumamoto, Japan), pentobarbital sodium (Tokyo Kasei Kogyo; Tokyo, Japan), BQ-485 and BQ-788 (Banyu Pharmaceuticals; Tsukuba, Japan), TAK-044 (Takeda Chemical Industries; Osaka, Japan). All other chemicals were of the highest analytic grade commercially available.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Concentration- and time-dependent effects of ET-1 on cell shortening and indo 1 fluorescence ratio. ET-1 applied by single administration to individual myocytes significantly increased the cell shortening at 3 × 10-11 M and higher concentrations, and the indo 1 ratio increased at 10-10 M and higher concentrations up to 10-9 M (Fig. 1). The increase in both signals in response to ET-1 up to 10-9 M developed gradually within 30 min; after the administration of ET-1 at 10-8 M, the steady level of cell shortening and indo 1 ratio was achieved within 15 min and remained stable up to 30 min. Actual tracings of cell shortening and indo 1 ratio after the administration of 10-9 M and 10-8 M are shown in Fig. 2, A and B, respectively.


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  		Fig. 1.   Time- and concentration-dependent changes in cell shortening and indo 1 fluorescence ratio in electrically stimulated rabbit single ventricular cardiomyocytes. Closed circles, cell shortening; open squares, indo 1 fluorescence ratio; vertical bars, means ± SE of 4-6 ventricular cardiomyocytes isolated from different rabbits; ordinate, the change in amplitude of cell shortening and indo 1 ratio expressed as percentage of the respective control value before the administration of endothelin (ET)-1 that was assigned to 100%; abscissa, time after administration of ET-1 in min. A: ET-1 10-11 M; B: ET-1 3×10-11 M; C: ET-1 10-10 M; D: ET-1 10-9 M; E: ET-1 10-8 M. *P < 0.05 vs. the respective control values at time 0.



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  		Fig. 2.   Actual tracings of the time-dependent effects of ET-1 on Ca2+ transients (indo 1 fluorescence ratio) and cell shortening in rabbit single ventricular myocytes loaded with indo 1-AM. A: ET-1 at 10-9 M; B: ET-1 at 10-8 M.

The cell shortening and indo 1 ratio were increased in a concentration-dependent manner by ET-1 up to 10-9 M (Figs. 1, A-D, and 2A). After the administration of ET-1 at 10-8 M the indo 1 ratio was not increased significantly compared with the level before the administration (107.7 ± 4.92%; n = 6). Instead a significant decrease in indo 1 ratio (87.9 ± 1.81%) and cell shortening (76.9 ± 1.88%) (n = 6, each; P < 0.05) was induced transiently (Figs. 1E and 2B). The increase in cell shortening in response to ET-1 at 10-8 M (150.3 ± 6.91%, n = 6) was significant compared with the control before the administration of ET-1 but was less than that induced by 10-9 M (208.4 ± 13.2%, n = 5, P < 0.05, Fig. 1).

The CRC for increases in cell shortening and indo 1 ratio in response to ET-1 was determined also by cumulative administration and summarized data are presented as the control response in Fig. 3, A and B, respectively. ET-1 elicited concentration-dependent increases in cell shortening and indo 1 ratio by cumulative administration, and the maximal response was achieved by ET-1 at 10-9 and 3 × 10-9 M (cell shortening, 179.6 ± 10.36%; indo 1 ratio, 150.4 ± 10.74%; n = 6, each). The EC50 values for cell shortening and indo 1 ratio were 8.6 × 10-11 and 4.6 × 10-11 M, respectively. The effects of ET-1 administered up to 3 × 10-9 M were not reversed by washout of ET-1 for 30 min.


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  		Fig. 3.   Influence of BQ-485, a selective ETA-receptor antagonist, on the cumulative concentration-response curves of ET-1 for increases in cell shortening (A) and indo 1 fluorescence ratio (B) in rabbit single ventricular cardiomyocytes. BQ-485 (10-7 M) was allowed to act for 20 min before and during exposure to ET-1. Closed symbols, control (n = 6 myocytes isolated from different animals); open symbols, in the presence of BQ-485 (n = 4 myocytes isolated from different animals); ordinate, changes in the amplitude expressed as percentage of the control that was assigned to 100%; abscissa, molar concentration of ET-1 on logarithmic scale; C, control values before the administration of ET-1. *P < 0.05 vs. the respective control values.

Influence of ETA-receptor blockade by BQ-485 on ET-1-induced effects. Influence of BQ-485, a selective ETA-receptor antagonist, on the ET-1-induced cell shortening and indo 1 ratio was investigated in two series of experiments.

In first series, the influence of 10-7 M BQ-485 on the CRCs for increases in cell shortening and indo 1 ratio induced by ET-1 was examined. BQ-485 at 10-7 M alone did not affect the cell shortening or indo 1 ratio (data not shown). The CRCs for increases in cell shortening and indo 1 ratio induced by ET-1 were shifted to the right, essentially in parallel 49- and 41-fold in the presence of 10-7 M BQ-485, when calculated graphically from mean CRCs at the level of EC50 (4.2 × 10-9 M for cell shortening; 1.9 × 10-9 M for indo 1 ratio) in Fig. 3. The maximum responses achieved by ET-1 (3 × 10-8 M) in the presence of BQ-485 were 187.9 ± 11.37% for cell shortening and 157.9 ± 11.35% for indo 1 ratio (Fig. 3, n = 4 each), which were not lower than those in control myocytes.

In second series, the influence of BQ-485 (10-7 M) on the effects of ET-1 by single administration was investigated. BQ-485 inhibited almost completely the effects of ET-1 at 10-10 M (Fig. 4A) and 10-9 M (Fig. 4B). By contrast, the effects of 10-8 M ET-1 on cell shortening (208.4 ± 13.2%) and indo 1 ratio (134.5 ± 10.3%) were significantly enhanced by the presence of BQ-485 (P < 0.05, n = 5, Fig. 4C) 30 min after the administration. Transient negative responses induced by 10-8 M ET-1 were abolished by BQ-485 (Fig. 4C).


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  		Fig. 4.   Influence of BQ-485 on the time- and concentration-dependent increases in cell shortening (circles) and indo 1 fluorescence ratio (squares) induced by ET-1 at 10-10 M (A), 10-9 M (B), and 10-8 M (C). Closed symbols, control (n = 5-6 myocytes isolated from different animals); open symbols, in the presence of BQ-485 at 10-7 M (n = 5-6 myocytes isolated from different animals); vertical bars, means ± SE of myocytes isolated from different rabbits; ordinate, changes in the amplitude expressed as percentage of the control that was assigned to 100%; abscissa, time after administration of ET-1 (in min). A: BQ-485 10-7 M on ET-1 10-10 M; B: BQ-485 10-7 M on ET-1 10-9 M; C: BQ-485 10-7 M on ET-1 10-8 M. *P < 0.05 vs. respective control values.

Influence of ETB-receptor blockade by BQ-788 on ET-1-induced effects. In this series, we examined the influence of a novel selective antagonist of ETB-receptors, BQ-788, on the ET-1-induced cell shortening and indo 1 ratio. BQ-788 at 10-6 M did not affect the baseline levels of cell shortening and indo 1 ratio (data not shown). BQ-788 did not affect the responses induced by ET-1 at 10-10 M (Fig. 5A); it partially inhibited the increases in cell shortening and indo 1 ratio induced by ET-1 at 10-9 M (Fig. 5B). The blockade of ETB-receptors abolished the initial inhibitory action of 10-8 M ET-1, and it enhanced the increases in cell shortening (175.6 ± 8.73%, P < 0.05) and indo 1 ratio (139.4 ± 3.59%, P < 0.05) induced by ET-1 at 10-8 M (n = 5 each), but the enhancement of cell shortening by BQ-788 (Fig. 5C) was less than that by BQ-485 (Fig. 4C).


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  		Fig. 5.   Influence of BQ-788, a selective ETB-receptor antagonist, on time- and concentration-dependent increases in cell shortening (circles) and indo 1 fluorescence ratio (squares) induced by ET-1 at 10-10 M (A), 10-9 M (B), and 10-8 M (C). Closed symbols, control (n = 4-6 myocytes isolated from different animals); open symbols, in the presence of BQ-788 at 10-6 M (n = 4-6 myocytes isolated from different animals); vertical bars, means ± SE of myocytes isolated from different rabbits; ordinate, changes in the amplitude expressed as percentage of the control that was assigned to 100%; abscissa, time after administration of ET-1 (in min). A: BQ-788 10-6 M on ET-1 10-10 M; B: BQ-788 10-6 M on ET-1 10-9 M; C: BQ-788 10-6 M on ET-1 10-8 M. *P < 0.05 vs. respective control values.

Influence of combined ETA- and ETB-receptor blockade by TAK-044. To examine the influence of combined blockade of both ETA- and ETB-receptors on the ET-1-induced responses, we investigated the influence of TAK-044, a nonselective ETA-/ETB-receptor antagonist, on cell shortening and indo 1 ratio induced by ET-1 at 10-8 M. In the presence of TAK-044 at 10-7 M, ET-1 at 10-8 M had no effects on the cell shortening and indo 1 ratio (Fig. 6).


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  		Fig. 6.   Influence of TAK-044, a nonselective ET-receptor antagonist, on the ET-1-induced changes in cell shortening (A) and indo 1 fluorescence ratio (B) in rabbit ventricular cardiomyocytes. TAK-044 (10-7 M) was allowed to act for 20 min before and during exposure to 10-8 M ET-1. Closed symbols, control (n = 6); open symbols, in the presence of TAK-044 (n = 4); ordinate, changes in the amplitude expressed as percentage of the control that was assigned to 100%; abscissa, time after administration of ET-1 (in min). *P < 0.05 vs. respective control values.

Comparison of contractile response to ET-1 and ET-3 in myocytes and papillary muscles. Whereas ET-1 increased cell shortening in single myocytes and induced PIE in isolated papillary muscles in a concentration-dependent manner, the former was more sensitive than the latter (Fig. 7A). The EC50 values calculated graphically from mean CRCs in Fig. 7A were 8.3 × 10-11 M in myocytes and 5.1 × 10-9 M in papillary muscles. Myocytes were therefore 62-fold more sensitive to ET-1 than papillary muscles. By contrast, the EC50 values for ET-3 were 1.1 × 10-8 M in myocytes and 8.5 × 10-9 M papillary muscles. There were no significant differences between the EC50 values derived for the effects of ET-3 on contractility of myocytes and papillary muscles (Fig. 7B).


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  		Fig. 7.   Comparison of the concentration dependence of contractile effects of ET-1 and ET-3 in rabbit single ventricular myocytes and rabbit isolated papillary muscles. A: ET-1. B: ET-3. Ordinate, changes in the amplitude of cell shortening (myocytes) or positive inotropic effect (papillary muscles) expressed as percentage of the maximal response of individual preparations that was assigned to 100%; abscissa, molar concentration of ET-1 or ET-3 on logarithmic scale. CRC for ET-3 in papillary muscles was taken from Ref. 19.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The important findings in the present study with ET-1- and ET-receptor antagonists in single rabbit ventricular myocytes are that: 1) single myocytes were more than 60-fold more sensitive to ET-1 in inducing the increase in cell shortening and Ca2+ transients than in producing the PIE in isolated rabbit papillary muscles; 2) in single myocytes a definite decrease in cell shortening (NIE) was elicited by ET-1 at 10-8 M; and 3) the PIE and NIE of ET-1 were susceptible to the ETA-receptor antagonist and also to the ETB-receptor antagonist.

Mechanisms responsible for the difference in ET-1-induced contractile response. Several potential factors may be involved in the mechanism underlying the difference in sensitivity to ET-1 between single myocytes and papillary muscles. First, ET-1 might be able to access more easily to the site of action in myocytes because of the absence of the endocardial endothelium. Provided that the diffusion of ET-1 is a limiting factor in papillary muscles, agents other than ET-1 might be affected in a similar manner. However, the extent of difference in the sensitivity of other agents was variable and not so great as that with ET-1. For example, the EC50 values for the agents, including ET-3 (myocytes vs. papillary muscles: 1.1 × 10-8 M vs. 8.5 × 10-9 M, Fig. 7B), angiotensin II (10-9 M vs. 1.1 × 10-8 M) and isoproterenol (1.70 × 10-9 M vs. 5.75 × 10-9 M) were not so greatly different between myocytes and papillary muscles (3, 7, 8, 23, 27). Furthermore, the treatment of papillary muscle with Triton X (0.1%), which has been demonstrated to cause selective damages to endocardial endothelial cells (15), did not appreciably affect the CRC for ET-1 in papillary muscles (1, 3).

The experimental temperatures used with myocytes and papillary muscles might contribute in part to the difference (6) because the experiments in myocytes were performed at room temperature, whereas those in papillary muscles were carried out at 37°C. However, it does not appear that the difference in temperature plays a crucial role because other agents, including ET-3, did not show a consistent alteration in the potency between myocytes and papillary muscles.

It is likely that the mechanism of action of the drug could be influenced by shortening or force development, because unloaded shortening was measured in single myocytes, whereas isometric force was measured in papillary muscles. Namely, single myocytes are not stretched properly but shorten from the slack length, whereas papillary muscles contract isometrically at muscle length near Lmax. To address this question, we compared the effect of levosimendan and angiotensin II in indo 1 loaded rabbit ventricular myocytes and aequorin-loaded papillary muscles. The concentration dependence of effects of levosimendan (22) and angiotensin II (8, 32) on contraction, Ca2+ transients, and Ca2+ sensitivity were identical in both preparations.

Another possibility is whether there could be a difference in the intracellular pH and hence Na+/H+ exchanger activity in myocytes compared with papillary muscles, i.e., thick papillary muscles (cross sectional area 0.79 mm2, diameter < 1.0 mm), stimulated at 1 Hz at 37°C, would be likely to have a hypoxic core, and hence, the myocytes in the central core may have a reduced pH. This could affect the response to ET-1 if modulation of intracellular pH by the Na+/H+ exchanger and secondary modulation of Na+/Ca2+ exchange are major mechanisms. Provided that this potential mechanism would play a crucial role in the difference in potency of ET-1, the effects of angiotensin II (10) and ET-3 (36) that act through an identical signal transduction process in respect to acceleration of the hydrolysis of phosphoinositides may be influenced in a similar manner to ET-1 in papillary muscles. However, because the effects of angiotensin II and ET-3 were not affected so greatly as ET-1 by the difference in preparations (8, 27), the difference in intracellular pH (pHi) would not play a crucial role. Furthermore, whereas the hypoxic core in papillary muscles could also influence the effect of isoproterenol, the CRC for isoproterenol was not markedly different between single myocytes and papillary muscles.

Although we did not carry out the experiments with ET-1 in the aequorin-loaded rabbit papillary muscles to elucidate the difference in relative contributions of the changes in the Ca2+ transient and myofilament Ca2+ sensitivity to the PIE of ET-1, angiotensin II showed identical effects on Ca2+ signals in myocytes (8) and papillary muscles (34).

In rabbit ventricular myocytes, the CRCs for the ET-1-induced increase in cell shortening and Ca2+ transients were shifted by the selective ETA-receptor antagonist BQ-485 to the right in parallel to a similar extent (Fig. 3). In contrast, in isolated rabbit papillary muscles, the CRC for PIE of ET-1 was biphasic. ET-1 up to 10-9 M induced the first phase [~20% of the maximal response to isoproterenol (Isomax)] and at 10-9 M and higher ET-1 induced the second phase that constituted the main portion of the CRC with the maximal response of 60% of Isomax (12, 24, 25). Because the first phase was susceptible for ETA antagonists, whereas the second phase was not antagonized but was rather enhanced by these antagonists (5, 12, 19), we supposed that the first phase in papillary muscles corresponds to the facilitatory response to ET-1 in myocytes and that the inhibitory substances may be released from cardiac and/or noncardiac cells in papillary muscles, which might be responsible for producing the low plateau of the first phase. According to this postulate, we pharmacologically examined the influence of potential endogenous inhibitory mechanisms by means of selective inhibitors, including NG-monomethyl-L-arginine (nitric oxide synthase inhibition), indomethacin (COX inhibition), atropine, 8-phenyltheophylline (adenosine-receptor antagonist), AF-DX 116 BS (muscarinic M2-receptor antagonist), and pertussis toxin (Gi protein inhibition) in preliminary experiments. However, these inhibitors did not affect appreciably the PIE of ET-1 in rabbit papillary muscles (3).

In summary, these findings indicate that: 1) the prominent difference in the contractile response to ET-1 between myocytes and papillary muscles was exerted selectively for ET-1; the difference occurs in ET-1 but not in ET-3, though they are structurally closely related and considered to act through an identical signal transduction pathway (5, 36); 2) the diffusion in papillary muscles is not a limiting factor; 3) the difference in experimental temperature does not play a crucial role; 4) the mode of contraction, i.e., isotonic and isometric, may not be essential; 5) difference in pH or presence of hypoxic core in papillary muscles does not play an important role; and 6) modulation by known cardiac inhibitory mechanisms examined is not responsible for the difference. Because the findings in the current study showed that the factors mentioned above may not play a key role, other factors, including the experimental procedure to prepare single myocytes and regulation of receptor sites on which ET-1 acts, may be responsible for producing the difference in the potency of ET-1 between myocytes and papillary muscles.

Cellular mechanisms for contractile regulation induced by ET-1. The PIE of ET-1 is due to increases in both the amplitude of Ca2+ transients and myofilament Ca2+ sensitivity (7, 31). The ET-1-induced increase in Ca2+ transients has been shown to be highly susceptible to the inhibition of Na+/Ca2+ exchanger induced by KB-R7943 (35), indicating that secondary activation of the exchanger subsequent to stimulation of Na+/H+ exchanger may play a crucial role in the ET-1-induced increase in Ca2+ mobilization (14). The ET-1-induced facilitation of L-type Ca2+ current may also contribute to the increase in contractile force and Ca2+ transients in rabbit ventricular myocardium (27, 33). Thus the increase in the amplitude of Ca2+ transients induced by ET-1 may be due to synergistic contribution of activation of L-type Ca2+ channels (27, 33) and Na+/Ca2+ exchanger (35), whereas activation of Na+/H+ exchanger may contribute to both the increase in Ca2+ transients and myofilament Ca2+ sensitivity induced by ET-1 in rabbit ventricular myocardium (30, 35). Although it is unknown how these mechanisms differ in myocytes and papillary muscles, it has been shown that angiotensin II that shares the signal transduction process with ET-1 to lead to an increase in Ca2+ transients and myofilament Ca2+ sensitivity elicited an identical action in indo 1 loaded myocytes (8) and aequorin-loaded papillary muscles (32).

Negative inotropic effect of ET-1. In single rabbit ventricular myocytes, the PIE of ET-1 at 10-8 M was counteracted by a pronounced secondary NIE that is responsible for the bell-shaped CRC of ET-1 in myocytes. The secondary NIE is not due to Ca2+ overload because it was associated with a suppression of the amplitude of Ca2+ transients (Fig. 1E). However, it is likely that the Ca2+ transient amplitude could still be suppressed under conditions of Ca2+ overload when spontaneous Ca2+ release from the sarcoplasmic reticulum (SR) could reduce the SR Ca2+ available for release in response to electrical stimulation. However, we have never observed any experimental evidence for diastolic Ca2+ release, such as an increase in diastolic levels of [Ca2+]i during the induction of NIE by ET-1. Considering that indo 1 is highly sensitive to detecting the alteration of the diastolic [Ca2+]i level, spontaneous Ca2+ release during diastole may be excluded.

Because the selective inhibition of ETA or ETB receptors enhanced the PIE of ET-1 at 10-8 M (Figs. 4 and 5), both subtypes are considered to be involved in the NIE. The observation that the selective ETA-receptor antagonist BQ-485 produced a parallel shift of the CRC for PIE of ET-1 indicates that the bell-shape CRC may be shifted to the right without altering the shape of the CRC. Although the Ca2+ transient amplitude was similar during the blockade of ETA and ETB receptors at 10-8 M ET-1, the percentage of cell shortening was higher with ETA blockade than with ETB blockade, an indication that each subtype may trigger different signaling pathways in addition to NIE mediated by both subtypes (Figs. 3 and 4). Although it is not clear whether the ETB blockade has a different effect on myofilament Ca2+ sensitivity than ETA blockade, we have observed in rabbit ventricular myocytes that the dual effect of ET-1 on calcium current (ICa) was abolished by the ETA antagonist FR-139317, whereas the inhibitory component of the ET-1-induced ICa response was selectively antagonized by the ETB antagonist BQ-788, which supports the postulate that ETA and ETB receptors may activate different signaling pathways in rabbit ventricular myocardium. The secondary NIE may be exerted by receptor-mediated suppression of Ca2+ mobilization because ET-receptor antagonists abolished both the decreases in cell shortening and Ca2+ transients. The secondary NIE of ET-1 is also induced in isolated rabbit papillary muscles, but the extent is much less compared with single myocytes (refer to Fig. 4 of Ref. 24 and Fig. 3 of Ref. 25). Furthermore, the second phase of the PIE of ET-1 was enhanced by selective antagonists of ETA receptors (5, 12, 19, 24), supporting the view that ET-1 causes the NIE in papillary muscles.

Patch-clamp experiments were carried out in single rabbit ventricular myocytes to elucidate the role of ICa in regulation of Ca2+ transients induced by ET-1 (33). ET-1 at 10-8 M elicited a biphasic effect, i.e., a long-lasting increase in ICa subsequent to a transient decrease (32). Whereas the decrease in ICa was inhibited by antagonists of ETA (FR-139317) or ETB (BQ-788) receptors, the increase in ICa induced by ET-1 was antagonized only by FR-139317 (33). Although the detailed analysis of the relationship between the concentration of ET-1- and ET-receptor antagonists was not carried out, it appears to be evident that the ET-1-induced inhibition of ICa is partially responsible for the decrease in Ca2+ transients, which leads to NIE in rabbit ventricular myocytes. Activation of protein kinase C (PKC) by tumor-promoting phorbol esters has been shown to lead to either PIE or NIE depending on the concentration applied and species of animals employed (4, 9, 26). In this context pHi altered by PKC via Na+/H+ exchanger might also be involved in the biphasic inotropic response to ET-1 (13, 14). Recently, we have examined the influence of the PKC inhibitor chelerythrine on the ET-1-induced biphasic response and found that chelerythrine inhibited the PIE leaving the NIE unaltered, an indication that the excessive PKC activation is unlikely to be responsible for the NIE (29).

In conclusion, contractile regulation by ET-1 in rabbit ventricular myocytes is quite different from that in papillary muscle. ET-1 was much more potent in myocytes than in papillary muscles inducing the contractile response. The effects of ET-1 on Ca2+ transients and cell shortening in single myocytes were effectively antagonized by the selective ETA-receptor antagonist BQ-485 and partially by the ETB-receptor antagonist BQ-788. These observations are essentially similar to those in other species including humans, rats, and guinea pigs (11, 16, 17, 21, 28). These findings imply that the anomalous pharmacological characteristics of the ET-1-induced PIE in rabbit papillary muscles (3, 12, 19) is not due to atypical nature of ET receptor subtypes in the rabbit heart. The integrated regulatory mechanisms in the multicellular preparation including noncardiac cells may be responsible for the resistance to ETA-receptor antagonists of the ET-1-induced PIE in rabbit papillary muscles.


    ACKNOWLEDGEMENTS

We thank T. Watanabe for sharing preparations of rabbit ventricular cardiomyocytes in some of the present experiments, and H. Sugawara for valuable technical advice in carrying out the experiments. We also thank Banyu Pharmaceutical (Tsukuba, Japan) for a generous supply of BQ-485 and BQ-788 and Takeda Chemical Industries (Osaka, Japan) for providing TAK-044.


    FOOTNOTES

This work was supported in part by Grants-in-Aid 11470021 and 11557203 for Scientific Research (B) from the Ministry of Education, Science, Sports and Culture (Japan) and by the Research Grant for Cardiovascular Disease (11C-1) from the Ministry of Health and Welfare (Japan).

Present address of M. A. H. Talukder: Dept. of Pharmacology, The Brody School of Medicine, East Carolina University, Greenville, NC 27858.

Address for reprint requests and other correspondence: M. Endoh, Dept. of Pharmacology, Yamagata University School of Medicine, 2-2-2 Iida-nishi, Yamagata 990-9585, Japan (E-mail: mendou{at}med.id.yamagata-u.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 30 October 2000; accepted in final form 12 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 281(2):H596-H605
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